The genomes of hundreds of bacteria have been sequenced, with even more to come. Yet, versatile genetic tools available for genome manipulations post-sequencing are far lacking behind. The proposed research is an initiation to solve some of these problems. With these available bacterial genomes and relentless occurrences of new emerging and reemerging infectious bacteria, existing universal genetic tools should be explored and modified further to increase the range of usefulness in these bacteria. The available universal genetic tools will allow studies to be performed on various bacterial pathogens that are serious burdens to public health. Thus, we are proposing to make significant improvements to two existing universal tools, namely, the promiscuous genetic transfer ability of bacterial RP4-based conjugation and the broadly applied Flp-FRT of the yeast 2-micron plasmid. More specifically, we will engineer several RP4-based conjugal donors that are suicidal post-conjugation. This will broaden the recipient-range of this universal system by alleviating the need for prior determination of appropriate selective media. In addition, such tools will be extremely useful in high-throughput genomic experiments, complementing other high-throughput technologies such as microarrays, genomic, and proteomics. The second aim is to engineer numerous useful constructs, containing unique FRTs (flip-recombination-target) of the yeast 2-micron plasmid, for bacterial genome manipulations. Developing universal genetic tools to study bacteria is our long-term goal, ', since this will aid in various research aspects of our laboratory and others, dealing with infectious diseases of bacteria. [unreadable] [unreadable] [unreadable]